Abstract
The licensing step of DNA double-strand break repair by homologous recombination entails resection of DNA ends to generate a single-stranded DNA template for assembly of the repair machinery consisting of the RAD51 recombinase and ancillary factors1. DNA end resection is mechanistically intricate and reliant on the tumour suppressor complex BRCA1–BARD1 (ref. 2). Specifically, three distinct nuclease entities—the 5′–3′ exonuclease EXO1 and heterodimeric complexes of the DNA endonuclease DNA2, with either the BLM or WRN helicase—act in synergy to execute the end resection process3. A major question concerns whether BRCA1–BARD1 directly regulates end resection. Here, using highly purified protein factors, we provide evidence that BRCA1–BARD1 physically interacts with EXO1, BLM and WRN. Importantly, with reconstituted biochemical systems and a single-molecule analytical tool, we show that BRCA1–BARD1 upregulates the activity of all three resection pathways. We also demonstrate that BRCA1 and BARD1 harbour stand-alone modules that contribute to the overall functionality of BRCA1–BARD1. Moreover, analysis of a BARD1 mutant impaired in DNA binding shows the importance of this BARD1 attribute in end resection, both in vitro and in cells. Thus, BRCA1–BARD1 enhances the efficiency of all three long-range DNA end resection pathways during homologous recombination in human cells.
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Data availability
Data supporting the findings of this study are available within the paper and its Supplementary Information. All reagents are available from the corresponding authors on reasonable request. Source data are provided with this paper.
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Acknowledgements
This study was supported by NIH award nos. R50 CA265315 (Y.K.), R01 GM141091 (W.Z.), R01 CA246807 (S.B.), R01 GM140127 (D.S.L.), R01 GM136717, R01 CA23728 (A.V.M.), F30 CA278370, T32 GM145432 (A.M.J.), R01 CA244212 and R01 NS119225 (A.A.H.), R01 CA219836 (D.Z.), R01 CA221858, R01 CA236606 and R35 GM118026 (E.C.G.), R00 GM140264 (E.V.W.), R01 GM115568 and R01 GM128731 (S.K.O.), R01 CA139429 (R.H.) and R01 CA168635, R01 ES007061 and R35 CA241801 (P.S.); by DOD award no. BC191160 (A.V.M.), ACS award nos. RSG-22-721675-01-DMC (W.Z.) and PF-22-034-01-DMC (C.M.R.) and CPRIT award nos. RP210102 (W.Z.), RP220269 (R.H.), RR200030 (S.K.O.), RR220068 (E.V.W.) and RR210023 (A.V.M.). S.B. is holder of the Mays Family Foundation Distinguished Chair in Oncology. P.S. is the holder of the Robert A. Welch Distinguished Chair in Chemistry (no. AQ-0012). Mass photometry analysis was performed at the Center for Innovative Drug Discovery and the Mays Cancer Center Drug Discovery and Structural Biology Shared Resource at University of Texas Health San Antonio, supported by CPRIT Core Facility Award no. RP210208 and National Cancer Institute Cancer Center Support Grant no. P30 CA054174. The Flow Cytometry Shared Resource at University of Texas Health San Antonio is supported by National Cancer Institute Cancer Center Support Grant no. P30 CA054174, a CPRIT Core Facility Award (no. RP210126) and an instrumentation grant from the National Institutes of Health (no. S10 OD030432). Cell biological images were generated at the Core Optical Imaging Facility at University of Texas Health San Antonio, supported by National Cancer Institute Cancer Center Support Grant no. P30 CA054174. We thank T. Rao for initial work on BRCA1–BARD1 DNA-binding fragments and V. A. Bohr for providing the WRN plasmid.
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S.S., J.M.D., N.T., H.K., C.X., Y.K., S.B., E.C.G. and P.S. conceived and designed experiments. S.S., J.M.D., N.T., H.K., C.X., A.M.J., C.M.R., V.B.R., W.L., R.M., Q.F., J.-H.J., S.Z. and A.B.S. performed experiments. W.L., Y.K., N.T., S.S., J.M.D., H.K., C.X., A.M.J., C.M.R., V.B.R., O.F., H.D. and B.M. created expression constructs and cell lines and purified proteins for the study. S.S., J.M.D., N.T., H.K., C.X., A.M.J., C.M.R., V.B.R. and R.M. analysed the data. S.S., J.M.D., P.S., S.B., E.C.G., Y.K., R.H., W.Z., A.A.H., A.V.M., E.V.W., S.K.O., D.S.L. and D.Z. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Influence of BRCA1-BARD1 on BLM and DNA2 activities.
a, SDS-PAGE analysis of BRCA1-BARD1 purified as described6. b, Mass photometry profile of BRCA1-BARD1, n = 3. c, Testing of BLM helicase mutant with BRCA1-BARD1. BRCA1-BARD1 was incubated with wild-type (8 nM) or the K695R (8 nM) variant of BLM and RPA (200 nM) with the 32P-labeled 2-kbp dsDNA substrate, n = 2. d, BRCA1-BARD1 (100 nM) was tested for its influence on ATP hydrolysis by BLM (10 nM). Following incubation of proteins with γ32P-ATP and unlabeled 2-kbp dsDNA (64 nM ends), reactions were analyzed by thin layer chromatography, n = 3. e, BRCA1-BARD1 was tested for its effect on the unwinding of a 2-kbp 32P-labeled dsDNA by Sgs1 (25 nM) and yRPA (200 nM) in a 20 min reaction. HD, heat-denatured DNA. Error bars show mean ± SEM; n = 3. f, BRCA1-BARD1 (20 nM) and DNA2 were incubated with 3′ Cy5-labeled Y DNA with and without RPA (20 nM) and then analyzed, n = 3. g, BRCA1-BARD1 (100 nM) was tested for its effect on ATP hydrolysis by DNA2 (100 nM). Reactions were analyzed following incubation of proteins with γ32P-ATP and unlabeled 2-kbp dsDNA (64 nM ends), n = 2. h, BRCA1-BARD1 (10 nM), BLM (1 nM), DNA2 (15 nM), RPA (200 nM) were incubated with the 32P-labeled 2-kbp dsDNA substrate. Reactions were subjected to heat denaturation prior to gel electrophoresis for 2 h followed by data quantification. Error bars show mean ± SEM; n = 3. i, dsDNA or j, ssDNA generated by heat denaturation of the dsDNA substrate was incubated with BLM (1 nM), DNA2 (15 nM), RPA (200 nM) with or without BRCA1-BARD1 (10 nM). Results were quantified and plotted. Error bars show mean ± SD; n = 3. In k, results from the 10 min and 15 min time points of experiments in panel i and j were graphed. n=number of independent experiments. Uncropped gel images are provided in Supplementary Fig. 1.
Extended Data Fig. 2 Characterization of WRN-DNA2 resection stimulation by BRCA1-BARD1.
a, His6-tagged BRCA1-BARD1 was tested for DNA2 interaction by affinity pulldown using Talon resin (specific for the His6 tag). The supernatant (S), wash (W) and SDS eluate (E) fractions were analyzed by SDS-PAGE and Coomassie blue staining, n = 2. b, c, Interaction between BRCA1-BARD1 and BLM fragments was assessed by affinity pulldown. Indicated GST-tagged BLM fragments immobilized on glutathione resin were incubated with BRCA1-BARD1. Proteins were eluted from the resin and analyzed by SDS-PAGE with Coomassie blue staining. S, supernatant and E, SDS eluate of resin, n = 2. d, Wild-type WRN (2 nM) or WRNK577A (2 nM) was tested with BRCA1-BARD1, RPA (200 nM), and the 2-kbp 32P-labeled dsDNA for helicase activity, n = 2. e, Effect of BRCA1-BARD1 (10 nM) on DNA end resection mediated by WRN (2 nM)-DNA2 (20 nM), RPA (200 nM), reaction mixtures were subjected to heat denaturation prior to gel electrophoresis for 2 h followed by data quantification. Error bars show mean ± SEM; n = 4. f, dsDNA or g, ssDNA generated by heat denaturation of the 2-kbp dsDNA substrate was incubated with WRN (2 nM), DNA2 (20 nM), RPA (200 nM) with or without BRCA1-BARD1 (10 nM) for the indicated times. Results from each dsDNA resection or ssDNA digestion experiment were quantified and plotted. Error bars show mean ± SD; n = 3. h, results from the 10 min and 15 min time points of experiments testing WRN-DNA2 from panel f and g were plotted as bar graphs. HD, heat-denatured DNA substrate. n=number of independent experiments. Uncropped gel images are provided in Supplementary Fig. 1.
Extended Data Fig. 3 Stimulation of EXO1 by BRCA1-BARD1.
a, BRCA1-BARD1 and EXO1 (5 nM) were incubated with 3′ 32P-labeled 80-mer dsDNA (2.5 nM) for 10 min and then analyzed. The results were quantified and plotted. Error bars show mean ± SEM; n = 3. b, Influence of BRCA1-BARD1 on removal of the 5′ nucleotide by EXO1 (5 nM). BRCA1-BARD1 and EXO1 were incubated with 5′ end labeled 30-mer dsDNA (2.5 nM) for 10 min and then analyzed. The results were quantified and plotted. Error bars show mean ± SEM; n = 3. c, BRCA1-BARD1 and yExo1 (5 nM) were incubated with 3′ 32P-labeled 80-mer dsDNA for 10 min and then analyzed. The results were quantified and plotted. Error bars show mean ± SEM; n = 3. d, e, BRCA1-BARD1 was tested with Lambda or T7 exonuclease (0.5 U) (NEB) using the 3′ 32P-labeled 80-mer dsDNA (2.5 nM) for 10 min and then analyzed. The results were quantified and plotted. Error bars show mean ± SEM; n = 3. n=number of independent experiments. Uncropped gel images are provided in Supplementary Fig. 1.
Extended Data Fig. 4 Single molecule analysis of Sgs1-Dna2.
a, Kymograph showing the movement of GFP-Sgs1 pre–bound to unlabeled dsDNA when chased with yeast Dna2, yeast RPA-mCherry (2 nM) and ATP with b, corresponding velocity distribution (N = 133;n = 3), and c, processivity plot (N = 133;n = 3). d, Kymograph showing the movement of GFP-Sgs1 pre-bound to unlabeled dsDNA when chased with yeast Dna2, yeast RPA-mCherry, BRCA1-BARD1 and ATP with e, corresponding velocity distribution (N = 69;n = 3) and f, processivity plot (N = 69;n = 3). g, Purified BRCA1-BARD1 with a C-terminal GFP tag on BARD1 was analyzed by SDS-PAGE. h, GFP-tagged BRCA1-BARD1 was incubated with BLM (5 nM), RPA (200 nM), and the 2-kbp 32P-labeled dsDNA substrate for 20 min. HD denotes heat-denatured DNA. i, Quantification of the results in panel h. Error bars show mean ± SEM from 3 experiments. j, Kymographs of DNA end resection by mCherry-BLM, GFP-DNA2, RPA and +/− BRCA1-BARD1. k, Velocity of resection reactions containing mCherry-BLM, GFP-DNA2, RPA and +/− BRCA1-BARD1. p < 0.0001; from two-tailed unpaired-t-test. l, Pie charts summarizing the localization pattern of mCherry-BLM and GFP-DNA2 from panel j. m, n, Binding distribution of GFP-EXO1 at 0 min with (N = 174; n = 3) and without BRCA1-BARD1 (N = 163; n = 3). Individual data points correspond to the dataset generated by bootstrap analysis; Error bars represent SD from the mean. N=number of DNA molecules analyzed; n=number of flow cell repeats. Uncropped gel images are provided in Supplementary Fig. 1.
Extended Data Fig. 5 BRCA1 and BARD1 DNA binding fragments and internal deletion mutants.
a, SDS-PAGE analysis of purified BARD1124–270. b, Testing of BARD1124–270 for DNA binding using Cy5-labeled 80 bp (3 nM) DNA as substrate. Results were quantified and plotted. Error bars show mean ± SEM; n = 3. c, SDS-PAGE analysis of purified BRCA1467–696. d, Testing of BRCA1467–696 for DNA binding using Cy5-labeled 80 bp (3 nM) DNA as substrate. Results were quantified and plotted. Error bars show mean ± SEM; n = 3. e, SDS-PAGE analysis of BRCA1-BARD1, BRCA1∆467–696-BARD1, BRCA1-BARD1∆124–270, and BRCA1∆467–696-BARD1∆124–270. f, Mass photometry profiles of wild type and mutant BRCA1-BARD1 species, n = 2. g, BRCA1-BARD1 and the indicated mutants were tested for DNA binding using Cy5-labeled 80 bp dsDNA (5 nM) as substrate. The reaction time was 10 min. h, Quantification of results from panel g, Error bars show mean ± SEM; n = 3. n=number of independent experiments. Uncropped gel images are provided in Supplementary Fig. 1.
Extended Data Fig. 6 Testing of BRCA1-BARD1 internal deletion mutants in DNA end resection.
a, Testing of BRCA1-BARD1 and the indicated mutants on the helicase activity of BLM (1 nM), RPA (200 nM) using the 32P-labeled 2-kbp dsDNA substrate (0.5 nM ends). The reaction time was 30 min. b, Quantification of results from panel a. Error bars show mean ± SEM; n = 3. c, Testing of BRCA1-BARD1 and the indicated mutants for their influence on DNA end resection mediated by BLM (1 nM), DNA2 (15 nM) and RPA (200 nM) using the 32P-labeled 2-kbp dsDNA substrate. The reaction time was 30 min. d, Quantification of results from panel c. Error bars show mean ± SEM; n = 3. e, Testing of BRCA1-BARD1 and the indicated mutants for their influence on DNA end resection mediated by EXO1 (5 nM) using the 3′ 32P-labeled 80-mer dsDNA (2.5 nM) substrate. f, Quantification of results from panel e. Error bars show mean ± SEM; n = 3. g, Testing of BRCA1-BARD1 and the indicated mutants on the helicase activity of WRN (2 nM), RPA (200 nM) using the 32P-labeled 2-kbp dsDNA substrate. The incubation time was 20 min. h, Quantification of results from panel g. Error bars show mean ± SEM; n = 5. n=number of independent experiments.
Extended Data Fig. 7 Testing of BRCA1 and BARD1 DNA binding domains on BLM and WRN helicase activity.
a, Testing of BRCA1467–696 and BARD1124–270 on the helicase activity of BLM (2 nM), RPA (200 nM) using the 32P-labeled 2-kbp dsDNA substrate. The reaction time was 30 min. b, Quantification of results from panel a. Error bars show mean ± SEM; n = 3. c, Testing of BRCA1467–696 and BARD1124–270 on the helicase activity of WRN (2 nM), RPA (200 nM) using the 32P-labeled 2-kbp dsDNA substrate. The incubation time was 20 min. d, Quantification of results from panel c. Error bars show mean ± SEM; n = 3. e, f, Interaction between BLM and BRCA1467–696 and BARD1124–270 was assessed by affinity pulldown (n = 2). MBP-BLM immobilized on amylose resin was incubated with BRCA1467–696 or BARD1124–270. Proteins were eluted from the resin and analyzed by SDS-PAGE with Coomassie blue staining. S, supernatant, W, wash and E, SDS eluate of resin. g, h, Interaction between GFP-WRN and BRCA1467–696 and BARD1124–270 was assessed by affinity pulldown assays (n = 2). GFP-WRN immobilized on anti-GFP resin was incubated with BRCA1467–696 and BARD1124–270. Proteins were eluted from the resin and analyzed by SDS-PAGE with Coomassie blue staining. S, supernatant, and E, SDS eluate of resin. n=number of independent experiments. Uncropped gel images are provided in Supplementary Fig. 1.
Extended Data Fig. 8 Construction and characterization of the BARD17KE DNA binding mutant.
a, Alignment of the human BARD1 DNA binding domain encompassing amino acid residues 124–270 with the equivalent region of the indicated BARD1 orthologues. Conserved residues with respect to human BARD1 are highlighted with yellow shade. Boxed lysine residues were changed in various combinations to glutamate to yield the 2KE, 3KE, 5KE, and 7KE mutants. b, SDS-PAGE analysis of BARD1WT and BARD1124–270 mutant species. c, Testing of the BARD1124–270 mutants for DNA binding using the Cy5-labeled 80 bp dsDNA (3 nM) substrate. d, Quantification of results from panel c. Error bars show mean ± SEM; n = 3. e, SDS-PAGE gel of purified BRCA1-BARD1 and BRCA1-BARD17KE. f, Profile of BRCA1-BARD1 and BRCA1-BARD17KE from mass photometry analysis, n = 2. g, Testing of BRCA1-BARD1 and BRCA1-BARD17KE using the a Cy5-labeled 80 bp dsDNA (5 nM) as substrate. h, Quantification of data from panel g. Error bars show mean ± SEM; n = 3. i, Testing BRCA1-BARD1 and BRCA1-BARD17KE for E3 ubiquitin ligase activity with histone H2A as substrate, n = 2. n=number of independent experiments. Uncropped gel images are provided in Supplementary Fig. 1.
Extended Data Fig. 9 Testing of BRCA1-BARD17KE with EXO1, WRN and WRN-DNA2.
a, BRCA1-BARD17KE was tested for interaction with EXO1. GFP-EXO1-FLAG and FLAG-BRCA1-BARD17KE. were incubated and EXO1 was captured using anti-GFP resin, n = 2. The supernatant and SDS eluate of the pulldown reaction were probed by immunoblotting for BRCA1 and EXO1 using anti-FLAG antibody. b, BRCA1-BARD17KE was tested for interaction with BLM, n = 2. FLAG-BRCA1-BARD17KE was preincubated with His6-BLM and captured using anti-FLAG resin. The supernatant and SDS eluate of the pulldown reaction were probed by immunoblotting for BRCA1 and BLM using anti-FLAG antibody and anti-His antibody. c, Testing of BRCA1-BARD1 and BRCA1-BARD17KE for their influence on the helicase activity of WRN (2 nM) with RPA (200 nM) and the 32P-labeled 2-kbp dsDNA substrate. The reaction time was 20 min. d, Quantification of results from panel c. Error bars show mean ± SEM; n = 3. e, Testing of BRCA1-BARD1 and BRCA1-BARD17KE DNA end resection mediated by WRN (2 nM), DNA2 (20 nM), and RPA (200 nM) using the 32P-labeled 2-kbp dsDNA substrate. f, Quantification of results from panel e. Error bars show mean ± SEM; n = 3. g, BRCA1-BARD17KE was tested for interaction with WRN, n = 2. BRCA1-BARD17KE was incubated with GFP-WRN and the latter was captured using anti-GFP resin. The supernatant and SDS eluate of the pulldown reaction were probed by immunoblotting for BARD1 and WRN using anti-BARD1 and anti-GFP antibody. n=number of independent experiments. Uncropped gel images are provided in Supplementary Fig. 1.
Extended Data Fig. 10 Cell biological characterization of BARD17KE cells.
a, Immunoblot analysis of lysates from HeLa cells with shRNA-mediated knockdown of BARD1 complemented with Myc-tagged BARD1WT or BARD17KE. b, Representative images of immunostaining of HeLa cells depleted of endogenous BARD1 by doxycycline treatment and expressing HA tagged BARD1WT or BARD17KE either mock-treated or exposed to ionizing radiation (6 Gy IR). Cells were pre-extracted and immunostained with anti-HA antibody (green), anti- γH2AX (red) and with DAPI (blue) to identify nuclei. Dot plot shows HA-BARD1 foci per cell (370, 349, 397 and 353 number of cells counted from left to right) from 3 independent experiments. Error bars show mean ± SEM;, ns=not significant. Scale bars, 10 μm. c, HeLa cells depleted of endogenous BARD1 by doxycycline treatment and expressing BARD1WT or BARD17KE were either mock-treated or exposed to ionizing radiation, and analyzed by single parameter flow cytometry after propidium iodide staining for DNA content (x-axis). See Supplementary fig. 2 for gating strategy. d, HeLa cells depleted of endogenous BARD1 and expressing BARD1 or BARD17KE were exposed to ionizing radiation and incubated for the indicated times. Cells were co-immunostained for 53BP1 foci (green) and cyclin A (to demarcate cells in S/G2, red). Nuclei were stained with DAPI (blue). e, Working model: our results demonstrate that BRCA1-BARD1 associates with DNA ends as well as with BLM, WRN, and EXO1 to accelerate long range resection. Mechanistically, we show that BRCA1-BARD1 translocates with the resection machinery and examination of various BRCA1-BARD1 mutants provides evidence that the DNA binding attribute of BRCA1-BARD1 is needed for efficient end resection by BLM-DNA2, WRN-DNA2, and EXO1. Schematic in e was created using BioRender (https://BioRender.com). Uncropped gel images are provided in Supplementary Fig. 1.
Supplementary information
Supplementary Information
This file contains Supplementary Figs. 1 and 2 (uncropped blots and gating strategy) and Table 1 (a list of oligonucleotides and antibodies used in the study).
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Salunkhe, S., Daley, J.M., Kaur, H. et al. Promotion of DNA end resection by BRCA1–BARD1 in homologous recombination. Nature (2024). https://doi.org/10.1038/s41586-024-07910-2
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DOI: https://doi.org/10.1038/s41586-024-07910-2
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